Figure 1.
Visualizing transport of the Tat substrate TorA-mCherry into the periplasmic space of E. coli cells.
(A) Fluorescence micrograph of E. coli BL21(DE3) cells expressing TorA-mCherry in the presence of the chromosomally encoded TatABC proteins. TorA-mCherry was expressed from a pET22b+-based plasmid (pPJ3) via the basal level of T7 RNA polymerase, which is synthesized in BL21(DE3) cells under non-inducing conditions and yet yields a sufficiently high fluorescence signal. Rim staining by TorA-mCherry (arrows) can be observed in most of the cells. Bar, 5 µm. (B) Expression of TorA-mCherry in the tatABCD deletion strain BL21(DE3) Δtat. The Tat-deficient cells are typically arranged in chains of non-separated cells and the fluorescent signal of TorA-mCherry is spread throughout the cytoplasm. (C) Expression of the non-transportable KK-variant of TorA-mCherry in tatABC wild type cells also leads to the retention of the fluorescent substrate in the cytosol.
Figure 2.
The TatA-GFP fusion is stable and functionally active.
(A) TatA-GFP was expressed in E. coli BL21(DE3) cells from a pBAD33 vector (pPR1) following 2 h of induction by the indicated amounts of arabinose. Whole cell proteins were precipitated with trichloroacetic acid and proteins were separated by SDS-PAGE. Shown is an immunoblot decorated with antibodies against TatA. TatABC wild type cells (wt) show a TatA signal at about 15 kDa (arrow) that is absent from tatABC deletion cells (Δtat) and enhanced in inner membrane vesicles (INV), which had been prepared from a TatABC-overexpressing strain. Cells induced with arabinose display a TatA-GFP band at about 36 kDa. Expression of TatA-GFP was slightly reduced by the co-expression of TorA-mCherry from plasmid pPJ3 (lanes 8 and 10). * non-specific band. (B–D) Expression of TorA-mCherry-SsrA from plasmid pPR8 in JARV16 (ΔtatAE) E. coli cells and the indicated transformants thereof. In the absence of TatAE, the Tat substrate remains cytosolic and cell separation is impaired (B). Transport of TorA-mCherry to the periplasm (arrows) and cell separation are restored by co-expression of TatA from plasmid pPR4 (C) and of TatA-GFP from plasmid pPR1 (D, inset shows cells from a different picture) demonstrating functionality of the TatA-GFP fusion. (E) Cells as in (D) now showing distribution of TatA-GFP. TatA-GFP forms similar clusters as when co-expressed with TatA (cf. Figure 3). (F) Growth of cell colonies on LB agar plates containing 2% SDS and 0.1% arabinose. Cells grown in LB liquid media were serially diluted and 5µl were each applied to the agar plate. Both TatA and TatA-GFP complement the growth defect of the ΔtatAE mutant strain JARV16 on 2% SDS. (G) Immunoblot of subcellular fractions decorated with antibodies against SufI. The strains MC4100 (wt), JARV16 (ΔtatAE) and JARV16/pPR1 (ΔtatAE+pBAD33TatA-GFP) were transformed with plasmid pPJ9 expressing the native Tat-substrate SufI. Cells were grown at 37° C and 180 rpm to an OD600 = 0.3 in LB-medium, supplemented with 0.1% L-arabinose for 1.5 h to induce synthesis of TatA-GFP and subsequently with 50 ng/mL anhydro-tetracycline for an additional 1.5 h to induce synthesis of SufI. Fractionation was carried out as described in Materials and Methods. Periplasmic fractions (P) and cytoplasm/membrane fractions (C/M) were loaded in a ratio 40:1 onto an 8% SDS-gel.
Figure 3.
Substrate-dependent clustering of TatA-GFP.
(A) E. coli BL21(DE3) wild type cells (wt) expressing TatA-GFP from plasmid pPR1. Expression was induced for 2h by the addition of 0.1% arabinose. The fluorescence signal of TatA-GFP is distributed evenly in the cell membrane with some tendency to cluster at the cell poles. (B) Clustering of TatA-GFP is totally missing in BL21(DE3) Δtat cells lacking a functional Tat-translocase (Δtat). TorA-mCherry co-expressed in the same cells from plasmid pPJ3 is not exported but remains cytosolic (lower panel). (C) Co-expression of TatA-GFP and TorA-mCherry in BL21(DE3) wild type cells (wt) harboring a functional Tat-translocase increases the number of TatA-GFP clusters while allowing transport of TorA-mCherry to the periplasm (inset). (D) Increased clustering of TatA-GFP in wild type cells is not observed when co-expressed with the non-transportable (cf. inset) KK-variant of TorA-mCherry from plasmid pPJ5.
Figure 4.
Quantification of TatA-GFP clusters.
Using the cell image analysis software CellProfiler as detailed in Materials and Methods, the average number of bright dots per 150 to 500 cells was each counted. Indicated are mean values with the standard errors of the means. Quantified were cells as depicted in Figure 3A (without substrate), 3C and 5A (+ RR-Tat substrate), 3D (+ KK-Tat substrate), 5B (+ RR-Tat substrate and 100 µM CCCP), and 5D (+ RR-Tat substrate after CCCP washout).
Figure 5.
PMF-dependent clustering of TatA-GFP.
(A) E. coli BL21(DE3) wild type cells (wt) co-expressing TatA-GFP from a pBAD33 vector (pPR1) and TorA-mCherry from a pET22 vector (pPJ3) show enhanced clustering of TatA-GFP as in Figure 3C. (B) A 30 min exposure of these cells to 100 µM of the protonophore CCCP impairs clustering in favor of a uniform distribution of TatA-GFP in the cell membrane (inset). (C–D) Removal of CCCP by washing the cells two times with fresh growth medium restores the clustering of TatA-GFP after 30 min and 180 min, respectively.
Figure 6.
In the presence of TatA and TatC, TatB localizes almost exclusively to the cell poles.
(A) E. coli BL21(DE3) wild type cells (wt) expressing TatB-mCherry from a pBAD33 vector (pPR2) following 2 h of induction with 0.1% arabinose. The fluorescence signal accumulates predominantly in polar foci with almost no staining of the cell bodies. (B) Co-expression of TatB-mCherry from a pBAD33 vector (pPR2) as in (A) and of the Tat substrate TorA-MalE335 from a pET22 vector (pPJ1) at non-induced levels. The additionally expressed Tat substrate does not change the staining pattern of TatB-mCherry. (C) Co-expression of TatB-mCherry and TorA-MalE335 in a BL21(DE3) Δtat cells. Without TatA and TatC (Δtat), TatB-mCherry is found dispersed in the cell periphery rendering the shape of the cells now more discernible against the background. (D) Equal expression levels of TatB-mCherry in cells depicted in (A–C). Whole cell proteins were precipitated with trichloroacetic acid and equivalent amounts were each subjected to SDS-PAGE. Immunoblots were decorated with antibodies against TatB and MalE as indicated. (E) Expression of TatB-mCherry in a ΔtatC strain (B1lK0). With TatC missing but native levels of TatB in the cells, TatB-mCherry is distributed evenly in the membrane as observed in a ΔtatABC strain (C). (F) When TatB-mCherry is expressed in a ΔtatB strain (BΦD) with endogenous TatC present, a polar localization of TatB-mCherry is observed as in a TatABC wild-type strain (A, B).
Figure 7.
Co-localization of TatA and TatB.
(A,B) TatA-GFP was expressed from a pQE-60 vector (pPR5) and TatB-mCherry from a pBAD33 vector (pPR2) in E. coli BL21(DE3) Δtat cells (Δtat). Cells were first induced with 0.1% arabinose for 1 h to start expression of TatB-mCherry and then for 1 h with 1 mM IPTG to additionally express TatA-GFP, before micrographs were taken. Without wild type TatABC, TatA-GFP (A) and TatB-mCherry (B) both localize uniformly to the cell periphery. (C–E) as (A,B) except that TatA-GFP and TatB-mCherry were co-expressed in tatABC wild type BL21(DE3) cells (wt). TatA-GFP (C) and TatB-mCherry (D) both form clusters, many of which coincide in the overlay (E).
Figure 8.
Expression of functional Tat substrates causes TatC to cluster at the cell poles.
(A) E. coli BL21(DE3) wild type cells (wt) expressing TatC-mCherry from a pBAD33 vector (pPR3). Expression was induced by the addition of 0.1% arabinose for 2 h. Overall weak fluorescent signal that in some cells is concentrated at the cell periphery (arrow). (B) Co-expression of TatC-mCherry from a pBAD33 vector (pPR3) as in (A) and of the Tat substrate TorA-MalE335 from a pET22 vector (pPJ1) at non-induced levels. The additionally expressed Tat substrate causes a pronounced clustering of TatC-mCherry at the cell poles. (C) Co-expression of TatC-mCherry from a pBAD33 vector (pPR3) as in (A) and of the natural Tat substrate SufI from a tightly controlled pASK-IBA33plus vector (pPJ9) by the subsequent addition of 50 ng/ml anhydro-tetracycline. Samples were taken at the indicated times and inspected under the microscope. Appearance of polar clusters starts after 30 min with a maximum reached after 50 min. No clustering of TatC-mCherry occurs without induction of SufI. (D) as in (B) expressing the non-functional KK-version of TorA-MalE335 from plasmid pPJ2. This does not lead to re-localization and clustering of TatC-mCherry at the poles. (E) as in (B) except that after inducing synthesis of TatC-mCherry and TorA-MalE335, 100 µM CCCP was added to the growth medium 30 min before cells were microscoped. Dissipation of the PMF does not interfere with the TorA-MalE335-dependent agglomeration of TatC-mCherry at the cell poles. (F) as in (B) except that TatC-mCherry and TorA-MalE335 were expressed in BL21(DE3) Δtat cells (Δtat). The TorA-MalE335-mediated clustering of TatC-mCherry at the cell poles is also independent of TatA and TatB. (G) Equal expression levels of TatC-mCherry in the cells depicted in the indicated panels. After 2 h of induction, whole cell proteins were precipitated with trichloroacetic acid and equivalent amounts were subjected to SDS-PAGE. Immunoblots were decorated with antibodies against TatC.